Peripheral Sarcoplasmic Reticulum Research Articles

Calcium/calmodulin-dependent kinase 2 mediates Epac-induced spontaneous transient outward currents in rat vascular smooth muscle.

Key points The Ca2+ and redox‐sensing enzyme Ca2+/calmodulin‐dependent kinase 2 (CaMKII) is a crucial and well‐established signalling molecule in the heart and brain.In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII.The vasodilator‐induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage‐dependent Ca2+ influx. How Epac activates STOCs is unknown.In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII.To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)‐sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+‐activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage‐dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8‐(4‐chloro‐phenylthio)‐2′‐O‐methyladenosine‐3′, 5‐cyclic monophosphate‐AM (8‐pCPT‐AM) induces autophosphorylation (activation) of calcium/calmodulin‐dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8‐pCPT‐AM‐induced increases in STOC activity. Epac‐induced CaMKII activation is probably initiated by inositol 1,4,5‐trisphosphate (IP3)‐mobilized Ca2+: 8‐pCPT‐AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8‐pCPT‐AM‐mediated CaMKII phosphorylation and STOC activity. 8‐pCPT‐AM does not directly activate BKCa channels, but STOCs cannot be generated by 8‐pCPT‐AM in the presence of ryanodine. Furthermore, exposure to 8‐pCPT‐AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine‐induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac‐mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox‐sensing enzyme in the regulation of vascular tone and blood flow.

Membrane potential and Ca2+ concentration dependence on pressure and vasoactive agents in arterial smooth muscle: A model.

Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these increases were reversed by NO.

Microtubules Couple Sarcoplasmic Reticulum Calcium Release to TRPM4 and BK Channel Activation in Cerebral Artery Myocytes

In cerebral artery myocytes, proximity of the sarcoplasmic reticulum (SR) and plasma membrane (PM) creates microdomains where Ca2+ released from the SR attains a concentration sufficient to activate large-conductance Ca2+-activated K+ (BK) and melastatin transient receptor potential 4 (TRPM4) channels; essential regulators of membrane excitability. Microtubules organize the SR in cardiac and skeletal myocytes, but it is not known if they serve this function in smooth muscle cells. Here, we test the hypothesis that microtubules maintain the SR architecture and Ca2+ microdomains essential for BK and TRPM4 activity. Using membrane- and tubulin-specific fluorescent dyes, we observed distinct microtubule arches beneath the peripheral SR proximal to the PM in contractile cerebral artery myocytes. Nocodazole, an inhibitor of microtubule polymerization, disrupted these subcellular structures and increased the SR to PM distance. Using high-speed, high-resolution confocal Ca2+ imaging, we found that microtubule depolymerization altered the spatiotemporal properties of localized Ca2+ signaling events. Nocodazole treatment also resulted in the loss of Ca2+-dependent TRPM4 and BK activity in perforated whole-cell patch clamp recordings and diminished contractility in pressure myography experiments. We conclude that the microtubules are essential for maintaining the SR architecture and Ca2+ microdomains necessary for the activation of BK and TRPM4 channels in cerebral artery myocytes. P01DK41315 (KMS) and R01HL091905 (SE).

Structural and Functional Arrangements of Atrial Myocytes that Facilitate Excitation-Contraction Coupling

Rabbit atrial myocytes lack transverse tubules and only the peripheral sarcoplasmic reticulum (SR) is in contact with the surface membrane (junctional SR; j-SR) and exposed to Ca influx through voltage-gated sarcolemmal Ca channels that initiates Ca release which propagates centripetally by Ca-induced Ca release from non-junctional SR (nj-SR). In this study we examined the structural and functional characteristics of excitation-contraction coupling (ECC) unique to atrial myocytes. Subsarcolemmal (SS) Ca release from j-SR preceded release at central (CT) nj-SR regions, and SS release was of higher amplitude compared to CT release. Using simultaneous measurements of cytosolic ([Ca]i) and intra-SR ([Ca]SR) Ca, we show that depletions of SS [Ca]SR were smaller compared to CT [Ca]SR depletions during electrical stimulation. Atrial myocytes revealed an approximately 1-2 µm wide gap devoid of mitochondria and SR structures between the j-SR and the nj-SR that the Ca signal must traverse to activate release from nj-SR. The spread of Ca from the j-SR to the nj-SR occurred very rapidly and was aided by the absence of mitochondria which might buffer the diffusion of Ca. In contrast to SS release, the continuation of Ca release through the nj-SR to the cell center occurred much slower in a Ca wave-like fashion, and the rise of [Ca]i detected at individual release sites of the nj-SR preceded the depletion of [Ca]SR. During this latency period a transient elevation of [Ca]SR was observed that acted as an intra-SR Ca sensitization wave that may facilitate the spread of excitation through the nj-SR via the luminal Ca-sensitivity of the ryanodine receptor Ca release channel. In summary, we have identified key structural and functional characteristics of atrial myocytes that allow for efficient ECC in the intact heart.

Atrial Excitation-Contraction Coupling and Ca Wave Propagation in Normal and Failing Hearts

We compared excitation-contraction coupling and Ca wave propagation in normal and heart failure (HF) rabbit atrial myocytes. Cytosolic Ca transients (Fluo-4 or Rhod-2), sarcoplasmic reticulum (SR) [Ca] (Fluo-5N) and mitochondrial localization (Mitotracker Red) were recorded by confocal microscopy. SEA and Ru360 were used to inhibit sarcolemmal Na/Ca exchange (NCX) or mitochondrial Ca uptake, respectively. In normal and HF rabbit atrial myocytes transverse tubules are missing, and the peripheral junctional SR (j-SR) is separated from the central non-junctional SR (nj-SR) by a 1-2 µm wide gap that is largely devoid of mitochondria and SR structures. During action potential (AP) stimulation SR Ca release initiated from the j-SR and propagated in centripetal direction via Ca-induced Ca release (CICR) from nj-SR. The centripetal Ca propagation velocity was highest across the gap between j-SR and nj-SR, slowed during propagation across the nj-SR and increased after Ru360 application. In HF, the centripetal propagation velocity was higher and mitochondrial density was decreased. Spontaneous Ca waves propagated with a leading wave front located to the cell periphery, i.e. arising from j-SR Ca release. Nearly half of all Ca waves in normal cells, but <10% in normal cells pretreated with SEA and none in ventricular cells triggered an AP, followed by a whole-cell Ca transient (termed here 'arrhythmogenic Ca waves'). The incidence of spontaneous Ca waves, the fraction of arrhythmogenic Ca waves and NCX activity were significantly increased in HF. In summary, the lack of mitochondria in the gap between j-SR and nj-SR enables rapid propagation of CICR from the j-SR to the nj-SR. Decreased mitochondrial Ca uptake in HF contributes to an increased centripetal Ca propagation velocity. Atrial myocytes are prone to develop NCX-dependent arrhythmogenic Ca waves, which is further accentuated in HF.

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca2+ transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) ‘Ca2+-release units’, which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.

Impact of sarcoplasmic reticulum calcium release on calcium dynamics and action potential morphology in human atrial myocytes: a computational study.

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca2+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.

Three‐dimensional electron microscopic reconstruction of intracellular organellar arrangements in vascular smooth muscle – further evidence of nanospaces and contacts

The sarcoplasmic reticulum (SR) of smooth muscle is crucial for appropriate regulation of Ca2+ signalling. In visceral and vascular smooth muscles the SR is known to periodically lie in close register, within a few nanometres, to the plasma membrane. Recent work has focussed on reconstructions of the ultrastructural arrangement of this so-called peripheral SR that may be important for the genesis of phenomena such as Ca2+ sparks. Here, we turn our attention to vascular smooth muscle and explore the 3-dimensional (3D) ultrastructural positioning of SR found deeper in the cell that is involved in the propagation of Ca2+ waves. We use digital reconstruction and volume rendering of serial electron microscopic sections from isolated resistance arteries, pressurized in vitro to mimic cellular geometric conformations anticipated in vivo, to map SR positioning. We confirm that these central portions of SR are in close register with mitochondria and the nucleus with all three organelles tightly enveloped by a myofilament/cytoskeletal lattice. Nanospacings between the SR and individual mitochondria are visible and in three dimensions as the SR contorts to accommodate these organelles. Direct connection of the SR and nuclear membranes is confirmed. Such 3D positioning of centrally located SR further informs us of its likely role in the manifestation of spatiotemporal Ca2+ dynamics: signal encoding may be facilitated by spatially directed release of Ca2+ to influence several processes crucial to vascular smooth muscle and resistance artery function including myofilament activation by Ca2+ waves, mitochondrial respiration and gene transcription.

Intracellular Diffusion Restrictions In Trout Cardiomyocytes

Rat cardiomyocytes are compartmentalized by barriers that restrict intracellular diffusion of adenine nucleotides. The exact localization of these diffusion barriers is unknown. Some possible candidates for diffusion restriction are t-tubules, sarcoplasmic reticulum (SR) and outer mitochondrial membrane. Further, rat cardiomyocytes have several parallel rows of mitochondria and myofilaments wrapped in SR, and it is possible that peripheral mitochondria and SR restrict diffusion to more central parts of the cell. Diffusion is facilitated by the creatine kinase system. Trout cardiomyocytes lack t-tubules and have a much more sparse SR. Additionally, single cardiomyocytes have only one layer of myofilaments surrounding a central core of mitochondria. We take advantage of the structural differences between rat and trout cardiomyocytes to study intracellular diffusion restrictions further. We measured the apparent ADP-affinity of trout skinned ventricular fibres at different temperatures to cover the physiological range for rainbow trout. Measurements were performed in the absence and presence of creatine to test whether diffusion is facilitated by the creatine kinase system. Our results show that trout cardiomyocytes are characterized by a low ADP-affinity. The affinity is temperature-dependent and increases with temperature. Creatine increases affinity at all temperatures, but the affinity in the presence of creatine is also temperature-dependent. The low ADP-affinity suggests that diffusion restrictions also exist in trout cardiomyocytes despite their structural difference with much more sparse membrane structures. This makes trout cardiomyocytes a useful model to study intracellular diffusion restrictions further.

A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle

Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.

Electron microscope tomography: further demonstration of nanocontacts between caveolae and smooth muscle sarcoplasmic reticulum

A spatial relationship between caveolae and sarcoplasmic reticulum (SR) in smooth muscle cells (SMC) was previously reported in computer-assisted three-dimensional reconstruction from transmission electron microscope serial sections. The knowledge of the three-dimensional organization of the cortical space of SMC is essential to understand caveolae function at the cellular level. Cellular tomography using transmission electron microscopy tomography (EMT) is the only available technology to reliably chart the inside of a cell and is therefore an essential technology in the study of organellar nanospatial relationships. Using EMT we further demonstrate here that caveolae and peripheral SR in visceral SMC build constantly spatial units, presumably responsible for a vectorial control of free Ca2+ cytoplasmic concentrations in definite nanospaces.

Caveolae and calcium handling, a review and a hypothesis

Caveolae are associated with molecules crucial for calcium handling. This review considers the roles of caveolae in calcium handling for smooth muscle and interstitial cells of Cajal (ICC). Structural studies showed that the plasma membrane calcium pump (PMCA), a sodium-calcium exchanger (NCX1), and a myogenic nNOS appear to be colocalized with caveolin I, the main constituent of these caveolae. Voltage dependent calcium channels (VDCC) are associated but not co-localized with caveolin 1, as are proteins of the peripheral sarcoplasmic reticulum (SR) such as calreticulin. Only the nNOS is absent from caveolin 1 knockout animals. Functional studies in calcium free media sugest that a source of calcium in tonic smooth muscles exists, partly sequestered from extracellular EGTA. This source supported sustained contractions to carbachol using VDCC and dependent on activity of the SERCA pump. This source is postulated to be caveolae, near peripheral SR. New evidence, presented here, suggests that a similar source exists in phasic smooth muscle of the intestine and its ICC. These results suggest that caveolae and peripheral SR are a functional unit recycling calcium through VDCC and controlling its local concentration. Calcium handling molecules associated with caveolae in smooth muscle and ICC were identified and their possible functions also reviewed.

Caveolar nanospaces in smooth muscle cells

Caveolae, specialized membrane nanodomains, have a key role in signaling processes, including calcium handling in smooth muscle cells (SMC). We explored the three-dimensional (3D) architecture of peripheral cytoplasmic space at the nanoscale level and the close spatial relationships between caveolae, sarcoplasmic reticulum (SR), and mitochondria, as ultrastructural basis for an excitation-contraction coupling system and, eventually, for excitation - transcription coupling. About 150 electron micrographs of SMC showed that superficial SR and peripheral mitochondria are rigorously located along the caveolar domains of plasma membrane, alternating with plasmalemmal dense plaques. Electron micrographs made on serial ultrathin sections were digitized, then computer-assisted organellar profiles were traced on images, and automatic 3D reconstruction was obtained using the ‘Reconstruct’ software. The reconstruction was made for 1 μm3 in rat stomach (muscularis mucosae) and 10 μm3 in rat urinary bladder (detrusor smooth muscle). The close appositions (about 15 nm distance) of caveolae, peripheral SR, and mitochondria create coherent cytoplasmic nanoscale subdomains. Apparently, 80% of caveolae establish close contacts with SR and about 10% establish close contacts with mitochondria in both types of SMC. Thus, our results show that caveolae and peripheral SR build Ca2+release units in which mitochondria often could play a part. The caveolae-SR couplings occupy 4.19% of the cellular volume in stomach and 3.10% in rat urinary bladder, while caveolae-mitochondria couplings occupy 3.66% and 3.17%, respectively. We conclude that there are strategic caveolae-SR or caveolae-mitochondria contacts at the nanoscale level in the cortical cytoplasm of SMC, presumably responsible for a vectorial control of free Ca2+ cytoplasmic concentrations in definite nanospaces. This may account for slective activation of specific Ca2+ signaling pathways.

On the Loose: Uncaging Ca2+-induced Ca2+ Release in Smooth Muscle

On the Loose: Uncaging Ca2+-induced Ca2+ Release in Smooth Muscle

Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: The relevance for Ca2+ oscillations and tone

A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action. Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca(2+) oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-beta-cyclodextrin (mbetacd), examined by light and electron microscopy. Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale-SR-myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR-caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mbetacd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca(2+) oscillations. We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca(2+) oscillations and contractile activation.

Ca2+ Regulation in the Near-Membrane Microenvironment in Smooth Muscle Cells

The microenvironment between the plasma membrane and the near-membrane sarcoplasmic reticulum (SR) may play an important role in Ca2+ regulation in smooth muscle cells. We used a three-dimensional mathematical model of Ca2+ diffusion and regulation and experimental measurements of SR Ca2+ uptake and the distribution of the SR in isolated smooth muscle cells to predict the extent that the near-membrane SR could load Ca2+ after the opening of single plasma membrane Ca2+ channels. We also modeled the effect of SR uptake on 1), single-channel Ca2+ transients in the near-membrane space; 2), the association of Ca2+ with Ca2+ buffers in this space; and 3), the amount of Ca2+ reaching the central cytoplasm of the cell. Our results indicate that, although single-channel Ca2+ transients could increase SR Ca2+ to a certain extent, SR Ca2+ uptake is not rapid enough to greatly affect the magnitude of these transients or their spread to the central cytoplasm unless the Ca2+ uptake rate of the peripheral SR is an order-of-magnitude higher than the mean rate derived from our experiments. Immunofluorescence imaging, however, did not reveal obvious differences in the density of SR Ca2+ pumps or phospholamban between the peripheral and central SR in smooth muscle cells.

Spatiotemporal characteristics of junctional and nonjunctional focal Ca2+ release in rat atrial myocytes.

Atrial myocytes have two functionally separate Ca2+ release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca2+ channels of surface membrane and those in central SR not associated with Ca2+ channels. Recently, we have reported on the gating of these two different Ca2+ release sites by Ca2+ current. In the present study, we report on the spatiotemporal properties of focal Ca2+ releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca2+ imaging. Peripheral and central sparks were similar in size and release time (approximately 300 000 Ca2+ ions for congruent with 12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd2+ and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had approximately 5-fold higher frequency, approximately 2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca2+ waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca2+ signaling diversity. The full text of this article is available at http://www.circresaha.org.

Localization of ryanodine receptors in smooth muscle.

The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide-stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR-Ca2+-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca2+ content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca2+-release channels and origins of Ca2+ sparks, Ca2+ oscillations, and Ca2+ waves.

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.

Agonist mobilization of sarcoplasmic reticular calcium in smooth muscle: functional coupling to the plasmalemmal Na +/Ca 2+ exchanger?

There is a close association of peripheral sarcoplasmic reticulum (SR), containing IP 3 receptors, and regions of the plasma membrane enriched in the Na +/Ca 2+ exchanger in smooth muscle. We have tested the possibility in rat uterine smooth muscle that Ca 2+ released from the SR is preferentially removed from the cytosol by the Na +/Ca 2+ exchanger. In Ca 2+-free solution, carbachol stimulation of myometria of non-pregnant rats resulted in transient increases in [Ca 2+] i and force due entirely to the release of SR Ca 2+. Inhibition of Na +/Ca 2+ exchange by removal of extracellular Na + did not alter the agonist-induced transients suggesting that Na +/Ca 2+ exchange was not involved in the removal of SR released Ca 2+. However, in myometria of pregnant rats, Na +/Ca 2+ exchange inhibition resulted in changes in the agonist-induced [Ca 2+] i transient profiles. The peak amplitude, duration and integral of carbachol-induced [Ca +] i transients were enhanced in Ca 2+-free/Na +-free solution without significantly affecting force transients. The lower rate of decay of [Ca 2+] i transients in Na +-free solution leads us to suggest that up to 35% of the SR released Ca 2+ may be extruded by the Na +/Ca 2+ exchanger in myometria of pregnant rats. Thus, in uterine smooth muscle, there is a gestational-dependent coupling of SR releasable Ca 2+ and plasmalemmal Na +/Ca 2+ exchange activity.